Peptide analogs capable of enhancing stimulation of a glioma-specific CTL response

ABSTRACT

The invention provides a peptide derived from the interleukin-13 receptor α2, which serves as a HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. The invention can be used as a vaccine for glioma and can be formulated into compositions for medical or veterinary use. In addition, the invention provides the use of a peptide derived from the Eph family of tyrosine kinase receptors which can be also used as a vaccine for glioma and can be formulated into compositions for medical or veterinary use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. patentapplication Ser. No. 11/231,618, which was filed on Sep. 21, 2005, nowU.S. Pat. No. 7,612,162, and which claims the benefit of U.S.Provisional Patent Application No. 60/611,797 filed Sep. 21, 2004, thedisclosure of both of which is incorporated by reference herein in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant NumbersCA117152 and NS040923 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotode/amino acid sequence listing submitted concurrently herewithand identified as follows: One 2,090 Byte ASCII (Text) file named“705268—ST25,” dated Jul. 2, 2010.

FIELD OF INVENTION

The invention pertains to reagents and methods for treatment of glioma.

BACKGROUND OF THE INVENTION

Brain tumors are particularly difficult to treat using conventionalmethods such as surgery, radiotherapy, or chemotherapy. Factors such asinvasive growth patterns and the blood-brain barrier make the treatmentof malignant gliomas more problematic than other tumors. The lack ofeffective treatment options for patients has led to the development ofalternative therapies, such as immunotherapy.

Immunotherapy is a promising new approach in the treatment of malignantgliomas. The efficacy of peripheral immunizations with autologous gliomacells or dendritic cells (DC) pulsed with synthetic peptides fortumor-antigen-specific T cell epitopes has been demonstrated inpreclinical mouse models (Okada et al., 2001; Okada et al., 1998).Specific T cell epitope-based vaccines are likely safer than wholeglioma cell-based vaccines due to the lack of theoretical autoimmuneresponses against normal brain components. Such antigen-specificapproaches may also be more effective than the bulk tumor-antigenapproaches because presentation of immunogenic T cell-epitopes andstimulation of antigen-specific T cell precursors can take place moreefficiently with the use of specific antigen-peptides than bulk tumorantigens.

The identification of T cell immuno-epitopes in human glioma associatedantigens is required for the development of such vaccines against humangliomas. Few cytotoxic T lymphocyte (CTL) immuno-epitopes have beenidentified for human malignant gliomas. However, an HLA (human leukocyteantigen)-A2-restricted cytotoxic T lymphocyte (CTL) epitope derived fromthe interleukin (IL)-13 receptor (R) α2 was recently identified (Okanoet al., 2002). IL-13Rα2 is known to be expressed in the majority ofhuman malignant gliomas but not in normal tissues (Debinski et al.,2000), thus making the identified epitope (IL-13Rα2₃₄₅₋₃₅₃) anattractive component of peptide-based vaccines for gliomas. Bygenerating unique CTL lines by stimulation of CD8+ cells with thepeptide IL-13Rα2₃₄₅₋₃₅₃, it was demonstrated that IL-13Rα2 positive,HLA-A2 positive glioma cells were efficiently lysed in anantigen-specific manner. However, it remains unclear how efficientlysuch peptide-based vaccines can induce specific CTLs and whetherpeptide-analogues can be used for optimal expansion and activation ofIL-13Rα2 specific HLA-A2-restricted CTL.

It has been demonstrated that certain amino acid substitutions inpeptides identified as CTL epitopes could greatly enhance the bindingaffinity of such peptides to the HLA (human leukocyte antigen) complexand thus would augment the immunogenicity of the peptide (Bownds et al.,2001; Chen et al., 2000). The enhancement of the immunogenicity ofIL-13Rα2₃₄₅₋₃₅₃, and other such epitopes could lead to the developmentof powerful, tumor-specific peptide-based vaccines, which would be asignificant improvement in the current treatment regime for malignantgliomas. However, there remains a need for an improved polypeptideHLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope.

As discussed above, few cytotoxic T lymphocyte (CTL) immuno-epitopeshave been identified for human malignant gliomas. Given the markedantigenic heterogeneity of gliomas, however, immunotherapy with a singletumor-specific T-cell epitope might merely promote transientstabilization of disease, prior to the progression of antigen lossvariants. EphA2 is a member of the Eph family of receptor tyrosinekinases, comprised of two major classes (EphA and EphB), which aredistinguished by their specificities for ligands (ephrin-A and ephrin-B,respectively). EphA2 is frequently overexpressed and often functionallydysregulated in advanced cancers, such as metastatic lesions (Kinch etal., 2003). Due to the aggressive and invasive nature of malignantgliomas, EphA2 might be expressed in this tumor entity and could be apotential target for glioma vaccines. T-cell immunoepitopes in EphA2have been identified and characterized as potential targets andsurrogate markers for other forms of cancer immunotherapy (Alves et al.,2003, and Tatsumi et al., 2003, the disclosures of which areincorporated by reference herein). The identification of additional CTLepitopes is a necessary step in the development of multiepitope-basedvaccines for glioma which would be a significant improvement in thecurrent treatment regime for malignant gliomas.

BRIEF SUMMARY OF THE INVENTION

The invention provides a peptide derived from IL-13Rα2, which serves asa HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. The inventivepeptide can comprise, consist of, or consist essentially of asubstitution mutant variant of WLPFGFILI (SEQ ID NO:1), where at leastone of the amino acid residues can be substituted for an amino acidother than the indicated residue. In addition, the inventive peptide cancomprise, consist of, or consist essentially of any of the followingsequences: WLPFGFILV (SEQ ID NO:2), ALPFGFILV (SEQ ID NO:3), orELPFGFILV (SEQ ID NO:4). The invention also provides a use of any of theabove peptides as a vaccine for glioma. In addition, the inventionprovides a method of vaccinating a patient against glioma, where thepeptide is introduced into a patient under conditions sufficient for thepatient to develop a CTL response. Further, the invention provides a useof a peptide having the sequence TLADFDPRV (SEQ ID NO:6) or acomposition comprising said peptide and a physiologically acceptablecarrier, as a vaccine for glioma. The invention also provides a methodof vaccinating a patient against glioma, where a peptide having thesequence TLADFDPRV (SEQ ID NO:6) or a composition comprising saidpeptide and a physiologically acceptable carrier, is introduced into apatient under conditions sufficient for the patient to develop a CTLresponse. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the accompanyingdrawings and the description of the invention provided herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 graphically presents data demonstrating that IL-13Rα2-V9 andIL-13Rα2-A1V9 induced a higher magnitude of CTL reactivity than thenative IL-13Rα2₃₄₅₋₃₅₃ or IL-13Rα2-E1V9 against T2 cells loaded withvarious concentrations of native IL-13Rα2₃₄₅₋₃₅₃. CD8+T cells from anHLA-A2+ glioma patient were stimulated with DCs loaded with eithernative IL-13Rα2₃₄₅₋₃₅₃ (●), IL-13Rα2-V9 (◯), IL-13Rα2-A1V9 (Δ),IL-13Rα2-E1V9 (X), Influenza M1₅₈₋₆₆ peptide (▾), or no peptide (□) for10 days. Then, the T cells were tested for lytic activity against T2cells loaded with indicated concentrations of IL-13Rα2₃₄₅₋₃₅₃ or nopeptide by 4-hr ⁵¹Cr-release assay. The E/T ratio was 12.5. P<0.01 forIL-13Rα2-V9 vs. native as well as IL-13Rα2-A1V9 vs. native at 0.1 and 1nM by two-tailed Student-t test. These data demonstrate results from oneof three separate experiments with similar results.

FIG. 2 graphically presents data demonstrating that the CTL line inducedby the V9 peptide had increased lytic activity against T2 cells loadedwith various concentrations of the wild type IL-13Rα2₃₄₅₋₃₅₃ peptide.The CTL lines induced by each of the 3 agonist analogues or the wildtype peptide were examined for CTL activities against lowerconcentrations of target IL-13Rα2₃₄₅₋₃₅₃ peptide with T2 cells loadedwith various concentrations (1-100 nM) of IL-13Rα2₃₄₅₋₃₅₃ by 4-Hr⁵¹Cr-release assay (E/T ratio=50).

FIG. 3 graphically presents data demonstrating that the modifiedpeptides induced a higher magnitude of CTL reactivity than the nativeIL-13Rα2₃₄₅₋₃₅₃ against human glioma cell lines. CD8+ cells derived froman HLA-A2+ glioma patient were stimulated with native IL-13Rα2₃₄₅₋₃₅₃(●), IL-13Rα2-V9 (◯), IL-13Rα2-A1V9 (Δ), or IL-13Rα2-E1V9 (X). On day10, the cells were tested for lytic ability against human glioma cellsSNB19 and U-251 (both are IL-13Rα+/HLA-A2+) using 4-Hr ⁵¹Cr-releaseassay. Against SNB19 glioma cells, p<0.05 at all E/T ratios forIL-13Rα2-V9 vs. native IL-13Rα2₃₄₅₋₃₅₃ as well as IL-13Rα2-A1V9 vs.native IL-13Rα2₃₄₅₋₃₅₃ by two-tailed Student-t tests. Against U251glioma cells, p<0.05 at E/T ratio of 10 and 40 for IL-13Rα2-V9 vs.native IL-13Rα2₃₄₅₋₃₅₃ as well as IL-13Rα2-A1V9 vs. nativeIL-13Rα2₃₄₅₋₃₅₃ by two-tailed Student-t tests. IL-13Rα2-E1V9 did notimprove the CTL reactivity for a statistically significant level incomparison to the native. The data presented represent one of threeexperiments with different donors with similar results.

FIG. 4 graphically presents data demonstrating that the addition of“cold” T2 cells pulsed with IL-13Rα2₃₄₅₋₃₅₃ inhibited the CTL activitiesindicating the antigen-specificity of the CTL lines. The CTL linesinduced with each peptide were incubated for 4 h with ⁵¹Cr-labeled humanglioma cell lines SNB19 at the indicated E:T ratios for evaluation ofspecific lytic ability (●). For the cold target inhibition assay,⁵¹Cr-labeled target SNB19 cells (1×10³ cells/well) and cold T2 cells(1×10⁴ cells/well) pulsed with (Δ) or without (◯) peptideIL-13Rα2₃₄₅₋₃₅₃ were incubated with the CTLs.

FIG. 5 graphically presents data demonstrating that the addition ofanti-HLA-A2 antibody inhibited the CTL activities indicatingHLA-A2-restricted recognition of the CTL lines. The CTL lines inducedwith each peptide were incubated for 4 h with ⁵¹Cr-labeled human gliomacell line SNB19 at the indicated E:T ratios for evaluation of specificlytic ability (●). Anti-HLA-A2 antibody (W6/32; 10 μg/ml) was added toblock the function of HLA-A2 mediated recognition by the T cells (◯).

FIG. 6 graphically presents data demonstrating that the modifiedpeptides induced higher magnitude of CTL reactivity than the nativeIL-13Rα2₃₄₅₋₃₅₃ against EL4-HHD loaded with the native IL-13Rα2₃₄₅₋₃₅₃.SPCs obtained from HHD mice that had been immunized with either controlMART-1₂₇₋₃₅ (●), native IL-13Rα2₃₄₅₋₃₅₃ (◯), IL-13Rα2-V9 (Δ) orIL-13Rα2-A1V9 (X) were tested for their specific lytic activity againstEL4-HHD cells pulsed with the native IL-13Rα2₃₄₅₋₃₅₃ by standard 4hr-⁵¹Cr-release assays.

FIG. 7 graphically presents data demonstrating that the modifiedpeptides induced a higher magnitude of CTL reactivity than the nativeIL-13Rα2₃₄₅₋₃₅₃ against EL4-HHD-IL-13Rα2. SPCs obtained from HHD micethat had been immunized with either control MART-1₂₇₋₃₅ (A), nativeIL-13Rα2₃₄₅₋₃₅₃ (B), IL-13Rα2-V9 (C), or IL-13Rα2-A1V9 (D) were testedtheir specific lytic activity against EL4-HHD-IL-13Rα2 (◯) or controlEL4-HHD (●) by standard 4 hr-⁵¹Cr-release assays.

FIG. 8 depicts the expression of EphA2 protein in glioblastomamultiforme (GBM) and anaplastic astrocytoma (AA). Paraffin embeddedsections of surgical specimens obtained from patients with GBM (A-C) orAA (D) were deparaffinized and stained with anti-EphA2 polyclonalantibody (C-20: Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), orcontrol rabbit IgG (upper right corner window for each sample).Relatively dense staining on endothelia and tumor cells surrounding thevessel was observed (D). Nine of fourteen GBM and six of nine AA casesexamined were positive for EphA2 (not shown). Original magnification;×20.

FIG. 9 graphically presents data demonstrating that the CD8+ cellsstimulated with EphA2₈₈₃₋₈₉₁ elicited CTL responses against human gliomacells expressing HLA-A2 and EphA2 protein. CD8+ T cells from an HLA-A2+glioma patients were stimulated with DCs loaded with EphA2₈₈₃₋₈₉₁ for 10days. These T cells were then tested for their lytic activity againsthuman glioma cells SNB19 (HLA-A2+, EphA2+), U251 (HLA-A2+, EphA2+) andA172 (HLA-A2−, EphA2+) by 4-hr ⁵¹Cr-release assay.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides an isolated peptide thatcomprises, consists of, or consists essentially of an amino acidsequence comprising, consisting of, or consisting essentially of asubstitution mutant variant of WLPFGFILI (SEQ ID NO:1), wherein at leastone of the amino acid residues in SEQ ID NO:1 is substituted with anamino acid other than the indicated residue. For example, at least two(e.g., three or more, four or more, etc.) of the amino acid residues inSEQ ID NO:1 can be substituted with an amino acid other than theindicated residue. Preferably, however, the inventive polypeptidecontains a substitution of only one or two of the amino acids. Moreover,while the inventive polypeptide can comprise such a sequence of aminoacids, more preferably, the inventive polypeptide consists essentiallyof such a sequence, and even more preferably, the inventive polypeptideconsists of such sequence (i.e., such that the inventive polypeptideconsists of nine amino acids).

The substitution from SEQ ID NO:1 can be, but need not be, aconservative substitution. Conservative substitutions are well known inthe art and can be amino acid replacements that preserve the structureand functional properties of proteins, such as the substitution of oneor more amino acids by similar amino acids. For example, a conservativesubstitution can be the substitution of an amino acid for another aminoacid within the same general class (e.g., an acidic amino acid, a basicamino acid, or a neutral amino acid).

Preferred embodiments of the inventive peptide include substituting theW in the first position of SEQ ID NO:1 with an amino acid other than W,substituting the I in the ninth position of SEQ ID NO:1 with an aminoacid other than I, or any combination thereof. For example, the W in thefirst position of SEQ ID NO:1 can be substituted with either A or E,and/or the I in the ninth position of SEQ ID NO:1 can be substitutedwith V. Preferred examples of the inventive peptide include isolatedpeptides comprising, consisting of, or consisting essentially of asequence of amino acid residues comprising, consisting of, or consistingessentially of WLPFGFILV (SEQ ID NO:2), ALPFGFILV (SEQ ID NO:3), orELPFGFILV (SEQ ID NO:4).

The inventive peptide can be prepared by methods known to those ofordinary skill in the art. For example, the inventive peptide can besynthesized using solid phase peptide synthesis techniques (e.g., Fmoc).Alternatively, the peptide can be synthesized using recombinant DNAtechnology (e.g., using bacterial or eukaryotic expression systems).Accordingly, to facilitate such methods, the invention provides geneticvectors (e.g., plasmids) comprising a sequence encoding the inventivepeptide, as well as host cells comprising such vectors. Methods forsolid state protein synthesis and recombinant protein synthesis arewell-known in the art. For example, “Molecular Cloning, A LaboratoryManual” (Sambrook et al., 3d Edition, Cold Spring Harbor Press), is awell-known reference detailing many suitable techniques for recombinantproduction of polypeptides. Accordingly, the invention provides theinventive peptide in recombinant form.

However it is made, the inventive peptide can be isolated and/orpurified (or substantially isolated and/or substantially purified).Accordingly, the invention provides the inventive peptide insubstantially isolated form (i.e., substantially isolated from otherpolypeptides or impurities). The peptide can be isolated from otherpeptides as a result of solid phase protein synthesis, for example.Alternatively, the peptide can be substantially isolated from otherproteins after cell lysis from recombinant production. Standard methodsof protein purification (e.g., HPLC) can be employed to substantiallypurify the inventive peptides. Thus, a preparation of the inventivepolypeptide preferably is at least 90% free of other polypeptides and/orcontaminants, and more preferably is at least about 95% free of otherpolypeptides and/or contaminants (such as at least about 97% or 98% freeof other polypeptides and/or contaminants). In a most preferredembodiment, the invention provides a preparation of the inventivepolypeptide that is greater than 99% free of other polypeptides and/orcontaminants (e.g., greater than 99.5% or even 99.9% or even 99.99% freeof other polypeptides).

In another embodiment, the invention provides a preparation of theinventive peptide in a number of formulations, depending on the desireduse. For example, where the peptide is substantially isolated (or evennearly completely isolated from other proteins), it can be formulated ina suitable medium solution for storage (e.g., under refrigeratedconditions or under frozen conditions). Such preparations can containprotective agents, such as buffers, preservatives, cryprotectants (e.g.,sugars such as trehalose), etc. The form of such preparations can besolutions, gels, etc., and the inventive peptide can, in someembodiments, be prepared in lyophilized form. Moreover, suchpreparations can include other desired agents, such as small moleculesor even other peptides and proteins, if desired. Indeed, the inventionprovides such a preparation comprising a mixture of differentembodiments of the inventive peptide (e.g., a plurality of peptidespecies as described herein). Technology for preparing such compositions(e.g., lyophilization, preparation of protein solutions, etc.), iswithin the state of the art.

In another embodiment, the invention provides a composition comprising,consisting of, or consisting essentially of one or more of the inventivepeptides (including mixtures thereof) and a physiologically acceptablecarrier or a pharmaceutically acceptable carrier. Any carrier which cansupply the peptide without destroying the vector within the carrier is asuitable carrier, and such carriers are well known in the art. Thecomposition can be introduced to a patient using any suitable methodwhich allows the patient to develop a CTL response. Such methods arewell known in the art and include, for example, parenteral, oral, andtopical administration. For example, a parenteral formulation couldconsist of a prompt or sustained release liquid preparation, dry powder,emulsion, suspension, or any other standard formulation. An oralformulation of the pharmaceutical composition could be, for example, aliquid solution, such as an effective amount of the compositiondissolved in diluents (e.g., water, saline, juice, etc.), suspensions inan appropriate liquid, or suitable emulsions. An oral formulation couldalso be delivered in tablet form, and could include excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. A topicalformulation could include compounds to enhance absorption or penetrationof the active ingredient through the skin or other affected areas, suchas dimethylsulfoxide and related analogs. The physiological orpharmaceutical composition could also be delivered topically using atransdermal device, such as a patch, which could include the compositionin a suitable solvent system with an adhesive system, such as an acrylicemulsion, and a polyester patch.

In addition to the inventive peptide carrier, as discussed above, theinventive composition can further comprise, consist of, or consistessentially of a T-helper epitope. Any suitable T-helper epitope whichstimulates an immune response, such as for example the stimulation ofCD4+ helper T cells and/or CD8+CTLs, can be used. In a preferredembodiment, the T-helper epitope can be or comprise a peptidecomprising, consisting of, or consisting essentially of a sequence ofamino acids comprising, consisting of, or consisting essentially ofTPPAYRPPNAPIL (SEQ ID NO:5).

In another embodiment, the invention provides a use of the inventivepeptide or composition as a prophylactic or therapeutic vaccine forglioma. The inventive peptide can be used to treat any type of glioma,such as ependymomas, astrocytomas, oligodendrogliomas, glioblastomas, ormixed gliomas, or prophylactically in the prevention of such diseases.Thus, the invention includes the use of the inventive peptide and/orcomposition for preparation of a medicament useful for vaccinating apatient against glioma.

The invention further provides a method of vaccinating a patient againstglioma, comprising, consisting of, or consisting essentially ofintroducing into the patient the inventive peptide or composition underconditions sufficient for said patient to develop a CTL response. Asnoted above, the composition can include a T-helper epitope, andinclusion of such epitope in the composition for use in the inventivemethod is preferred, but not necessary.

In another embodiment, the invention provides a use of a peptide having(e.g., comprising, consisting essentially of, or consisting of) theamino acid sequence TLADFDPRV (SEQ ID NO:6) as a prophylactic ortherapeutic vaccine for glioma. The invention also provides a use of acomposition comprising, consisting of, or consisting essentially of apeptide having the amino acid sequence TLADFDPRV (SEQ ID NO:6) and aphysiologically acceptable carrier such as a prophylactic or therapeuticvaccine. Thus, the invention provides the use of such peptide forpreparation of a medicament useful for vaccinating a patient againstglioma.

This aspect of the invention further provides a method of vaccinating apatient against glioma, comprising, consisting of, or consistingessentially of introducing into the patient a peptide having (e.g.,comprising, consisting essentially of, or consisting of) the amino acidsequence TLADFDPRV (SEQ ID NO:6) under conditions sufficient for saidpatient to develop a CTL response. In another embodiment, the inventionprovides a method of vaccinating a patient against glioma, comprising,consisting of, or consisting essentially of introducing into the patienta composition comprising, consisting of, or consisting essentially of apeptide having the sequence TLADFDPRV (SEQ ID NO:6) and aphysiologically acceptable carrier. The physiological carrier can be apharmaceutically acceptable carrier. As discussed above, any carrierwhich can supply the peptide without destroying the vector within thecarrier is a suitable carrier, and such carriers are well known in theart. The composition can include a T-helper epitope, as noted above, andinclusion of such epitope in the composition for use in the inventivemethod is preferred, but not necessary. The peptide can be used to treatany type of glioma, such as ependymomas, astrocytomas,oligodendrogliomas, glioblastomas, or mixed gliomas, or prophylacticallyin the prevention of such diseases.

In accordance with the inventive method, regardless of the exact peptideused or the formulation of the composition, the patient can be anyindividual that has been diagnosed with glioma or is identified as atrisk for developing glioma. In a preferred embodiment, the patient is amammal. Even more preferably, the patient is human.

In accordance with the inventive method, the peptide or composition canbe introduced to the patient by any suitable method, such as thosedescribed above. For therapeutic use, the peptide preferably isintroduced locally into the situs of the glioma or systemically inamounts sufficient to treat the glioma. For prophylactic use, thepeptide or composition is introduced into a patient in any suitablemanner to deliver a sufficient amount of the protein to the patient toachieve a protective effect.

For therapeutic use, following introduction of the peptide orcomposition into the patient, in accordance with the inventive method,the patient's condition is monitored to assess the severity of theglioma. Suitable application of the inventive method will result inslowing of the progression of the glioma and, in preferred embodiments,result in plateauing of the progress of the disease. Indeed, in morepreferred embodiments, application of the inventive method will resultin shrinkage of glioma in the patient or even in substantial or completeremission of the glioma. Thus, while the inventive method can lead to acure of the glioma in some patients, any degree of improvement in theprognosis of the patient following application of the inventive methodis considered to be successful application. Moreover, it is to beunderstood that the inventive method can be used as monotherapy oradjunctively in combination with other therapeutic agents (e.g.,chemotherapy or radiotherapy) or therapeutic methods.

For prophylaxis, following introduction of the inventive polypeptide orcomposition into the patient, the patient is suitably monitored toassess the development of glioma and/or continued risk of developingglioma. Successful prophylaxis can be measured by the absence of gliomain the patient for longer than the initial assessment of risk hadpredicted.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope. Briefly, theexamples demonstrate the generation of a potent agonist analogue peptideidentified here, which can induce IL-13Rα2-specific CTL responses evenmore efficiently than the original IL-13Rα2₃₄₅₋₃₅₃. ThreeIL-13Rα2₃₄₅₋₃₅₃ analogue peptides have been created by substituting thecarboxy-terminal isoleucine (I) for valine (V) and the amino terminaltryptophan (W) for either alanine (A), glutamic acid (E), ornon-substituted (W) (designated as A1V9, E1V9 and V9, respectively).Relative immunogenicity of these IL-13Ralpha2 (345-353) peptideanalogues was examined by stimulating peripheral blood cells (PBL) fromglioma patients with dendritic cells (DC) loaded with each of thesepeptides. Among the peptide analogues tested, V9 was capable of inducingIL-13Rα2₃₄₅₋₃₅₃ specific CTL from PBL of glioma patients moreefficiently than IL-13Rα2₃₄₅₋₃₅₃ consistently in all donors. Thesefindings suggest that a highly antigenic IL-13Rα2 peptide-analogue V9will be useful for the development of vaccines capable of expandingIL-13Rα2 specific CTL in glioma patients. The following examples alsodemonstrate the ability of the peptide EphA2₈₈₃₋₈₉₁ to induce an EphA2specific CTL response, thus suggesting that the peptide would be usefulin the development of additional vaccines for glioma.

Example 1

This example demonstrates the identification of modified peptides forIL-13Rα2₃₄₅₋₃₅₃ that enhance induction of the CTL response againstnative IL-13Rα2₃₄₅₋₃₅₃.

Three modified peptides were synthesized as listed in Table 1. Thebinding capability of these modified peptides was assessed using anHLA-A2 transfected T2 cell line. Aliquots of T2 cells were incubatedwith modified peptides or IL-13Rα2₃₄₅₋₃₅₃ at 1 nM overnight, and thenexamined for the surface expression levels of HLA-A2 on T2 cells by flowcytometry. Since stable binding of HLA-A2 with peptide epitopes furtherstabilizes the surface expression of HLA-A2 (Francini et al., 2002;Alves et al., 2003), quantitative expression levels of HLA-A2, which isindicated by Mean Fluorescence Intensity (MFI) in Table 1, correlatewith the binding affinity of the peptide-epitopes that are co-incubatedwith the T2 cells. The modified peptides V9 and A1V9 possess higherbinging affinity to HLA-A2 than the native IL-13Rα2₃₄₅₋₃₅₃ (Table 1),suggesting the possibility that these modified peptides are moreimmunogenic than the IL-13Rα2₃₄₅₋₃₅₃.

Peptide Amino Acid Sequence Binding Index(MFI*) Designation WLPFGFILI(SEQ ID NO: 1) native IL-13Rα2₃₄₅₋₃₅₃ 237.4 native WLPFGFILV (SEQ ID NO:2) V9: I was replaced with V at P9 375.6 V9 ALPFGFILV (SEQ ID NO: 3)A1V9: W→A at P1, and I→V at P9 462.8 A1V9 ELPFGFILV (SEQ ID NO: 4) E1V9:W→E at P1, and I→V at P9 241.6 E1V9 (Control: Non peptide) 121.8 *MeanFluorescence Intensity at the peptide concentration of 1 nM

Example 2

This example demonstrates that CTL induced by the agonist analogue V9recognized peptide IL-13Rα2₃₄₅₋₃₅₃ presented on HLA-A*0201 moreefficiently than CTL induced by the wild type peptide.

Dendritic cells (DCs) derived from HLA-A*0201⁺ glioma patients werepulsed with either V9, A1V9, E1V9, a control influenza (flu), or thewild type peptide (10 μg/ml), and used to stimulate autologous CD8⁺ Tcells. On day 7, the individual responder cell cultures were thenrestimulated once with autologous DCs loaded with the correspondingpeptide used in the primary stimulation. Specific CTL activity of theinduced T cell lines was first tested with T2 cells loaded with the wildtype IL-13Rα2₃₄₅₋₃₅₃, or no peptide on day 10.

As depicted in FIG. 1, the T cells that had been stimulated with eitherwild type (IL-13R) or agonist analogues (V9, A1V9 and E1V9) efficientlylysed T2 target cells pulsed with 100 ng/ml wild type IL-13Rα2₃₄₅₋₃₅₃;whereas only low background lysis was observed in the absence of thepeptide on T2 cells. T cells that had been stimulated with the controlflu-peptide or no-peptide (control) did not demonstrate any lyticactivity over background levels. These results demonstrated that the CTLlines induced with the wild type or agonist analogues recognized andlysed the cells presenting wild type IL-13Rα2₃₄₅₋₃₅₃ epitopespecifically. In particular, the V9 peptide induced a significantlyhigher level of antigen-specific CTL response in comparison to the wildtype IL-13Rα2₃₄₅₋₃₅₃ at each effector/target (E/T) ratio (p=0.018, 0.020and 0.011 at an E/T ratio of 50, 25 and 12.5, respectively). The sameset of experiments were repeated with at least three individual HLA-A2+glioma patients, and the V9 peptide consistently demonstrated higher CTLactivities than the native IL-13Rα2₃₄₅₋₃₅₃ in all four donors tested(data not shown).

Subsequently, the sensitivity of the CTL lines induced by agonistanalogues or the wild type peptide was examined with T2 cells loadedwith various concentrations (1-100 nM) of the IL-13Rα2₃₄₅₋₃₅₃ peptide by4-Hr ⁵¹Cr-release assay (FIG. 2). All CTL lines demonstratedpeptide-dose dependent lytic activities against peptide-loaded T2 cells.The CTL line induced by the agonist analogue V9 demonstrated higher CTLactivities than the wild type IL-13Rα2₃₄₅₋₃₅₃ at allpeptide-concentrations examined (P=0.029, 0.039 and 0.018 at 1, 10 and100 mM, respectively). It is noteworthy that the average percent lysisvalue achieved by V9-induced CTL with 1 nM IL-13Rα2₃₄₅₋₃₅₃ was higherthan that demonstrated with wild type peptide-induced CTL with 100 nMpeptide, although this did not demonstrate a statistical significancedue to a large standard variation. These results indicate that the V9peptide is more efficient than the wild type peptide in inducing CTLthat are capable of recognizing low concentrations of the target wildtype IL-13Rα2₃₄₅₋₃₅₃ peptide. This ability is important because humantumor cells express low levels of target CTL epitopes on theirHLA-molecules (Bakker et al., 1995; Lupetti et al., 1998).

Example 3

This example demonstrates that CTL induced by modified peptides lysedHLA-A2+ glioma cells that express IL-13 Rα2 more efficiently than CTLinduced by the native peptide.

The ability of modified peptides, such as IL-13Rα2-V9, to enhance theCTL activity against HLA-A2+ human glioma cells that endogenouslyexpressed and presented IL-13Rα2-derived epitopes was examined. Humanglioma cell lines U251 and SNB19 express HLA-A2 and IL-13Rα2, whereashuman glioma cell line A172 expresses IL-13Rα2 but not HLA-A2 (Okano etal., 2002). Therefore, U251 and SNB19 were used as relevant targetglioma cells, while A172 served as a negative control line todemonstrate HLA-A2-restriction of the response.

The lytic ability of the peptide-induced CTL lines against these gliomacells was examined using 4-hr ⁵¹Cr-release assays. As illustrated inFIG. 3, the U-251 and SNB19 cell lines were highly susceptible tocytotoxic activity of all CTL cell lines that had been induced withIL-13Rα2₃₄₅₋₃₅₃ or each of its modified peptides. A172 cells, incontrast, were not lysed beyond the background level (<10%) by any ofthe CTL lines tested, suggesting that the IL-13Rα2₃₄₅₋₃₅₃ or modifiedpeptide-induced CTL lines lysed SNB19 and U-251 glioma cells in anHLA-A2 restricted manner (data not shown). The T cells stimulated with amelanoma associated antigen epitope Mart-1 (27-35) and T cells with nopeptide stimulation showed only background level (<10%) lysis at allEffector/Target (E/T) ratios tested (data not shown). In this particularpatient, both IL-13Rα2-V9 and -A1V9 induced higher levels of lysis ofSNB19 and U-251 in each E/T ratio in comparison to the nativeIL-13Rα2₃₄₅₋₃₅₃ peptide.

To determine the specificity of the lytic activity, cold targetcompetition experiments were performed by addition of non-radiolabeled(cold) T2 cells pulsed with IL-13Rα2₃₄₅₋₃₅₃ peptide in the 4-h⁵¹Cr-release assay (FIG. 4). The anti-SNB19 glioma cell lytic activitiesby the CTL lines induced by the native IL-13Rα2₃₄₅₋₃₅₃ or IL-13Rα2-V9were almost completely inhibited by the addition of the cold T2 cellspulsed IL-13Rα2₃₄₅₋₃₅₃. The CTL activities, however, were not inhibitedby the addition of non-peptide pulsed cold T2 cells, demonstrating thatthe lytic ability of the CTLs was specific for the epitopeIL-13Rα2₃₄₅₋₃₅₃.

Furthermore, anti-HLA-A2 antibody (W6/32) was used to block the HLA-A2mediated signaling in the CTL reactivity. As illustrated in FIG. 5,addition of this antibody inhibited the CTL-mediated lysis, confirmingthat the anti-glioma CTL reactivity induced by these peptides was HLA-A2restricted.

Example 4

This example demonstrates the vaccination of HLA-A2 transgenic (HHD)mice with IL-13Rα2-derived CTL epitopes.

In order to examine whether immunization with IL-13Rα2₃₄₅₋₃₅₃ and/or itsmodified peptides can elicit CTL responses in vivo, and also to examinewhether induced CTL responses can mediate therapeutic anti-tumorresponses against IL-13Rα2₃₄₅₋₃₅₃-expressing brain tumors, the HHD micewere obtained from Dr. Francois A. Lemonnier (Pasteur Institute, Paris).HHD mice are D^(b)×β2 microglobulin (β2M) null, and transgenic formodified HLA-A2.1-β2 microglobulin single chain (HHD gene) (Pascolo etal., 1997). In vivo experiments showed that HHD mice exhibitHLA-A2-restricted responses to multiepitope proteins such as intactinfluenza virus (Pascolo et al., 1997) and novel cancer associatedantigens, such as EphA2 (Alves et al., 2003), HER-2/neu and hTERT(Scardino et al., 2002), MAGE (Graff-Dubois et al., 2002) and a novelbreast carcinoma associated BA46 (Carmon et al., 2002). Hence, thesemice are a useful tool for the identification and characterization ofpotential tumor-derived, HLA-A2-restricted CTL epitopes.

To create an HHD mouse-syngeneic tumor cell line that expressesIL-13Rα2, HHD gene-transfected EL4 lymphoma cells (EL4-HHD) wereobtained. EL4-HHD cells have been generated from EL4 by depletion ofD^(b)×β2M and insertion of modified HLA-A2.1-β2M single chain (Pascoloet al., 1997), thereby allowing syngeneic transplantation in HHD mice.EL4-HHD cells were stably transfected with an expression plasmidencoding IL-13 Rα2. The cell line (EL4-HHD-IL-13Rα2) expressed IL-13Rα2protein and formed tumors both in subcutaneous (s.c.) and intracranial(i.c.) space following injections to syngeneic HHD mice.

Example 5

This example demonstrates that in vivo immunization of HHD mice with themodified peptides induced higher magnitudes of CTL responses than thenative peptide against the target cells expressing IL-13Rα2₃₄₅₋₃₅₃.

HHD mice received (on days 7 and 14) s.c. injections of 100 μg ofpeptide IL-13Rα2-V9, -A1V9, IL-13Rα2₃₄₅₋₃₅₃, or MART-1₂₇₋₃₅ emulsifiedin incomplete Freund's adjuvant (IFA) in the presence of 140 μg of theI-A^(b)-restricted HBVcore₁₂₈ (TPPAYRPPNAPIL) (SEQ ID NO:5) T-helperepitope, which stimulates a CD4+ helper T cell response, therebypromoting the stimulation of CD8+CTLs. Control animals received IFAcontaining HBV helper-peptide only. Eleven days after the lastimmunization, the animals were sacrificed, and 5×10 spleen cells (SPCs)were stimulated in vitro with the same peptide that was used for in vivostimulation (10 μM). On day 6 of culture, the bulk populations weretested for specific cytotoxicity against the EL4-HHD cells expressingIL-13Rα2 or EL4-HHD pulsed with IL-13Rα2_(345-353.)

EL4-HHD-IL-13Rα2 and EL4-HHD were labeled with 100 μCi of ⁵¹Cr for 60min, plated in 96-well V-bottomed plates (3×10³ cell/well). LabeledEL4-HHD were pulsed with IL-13Rα2₃₄₅₋₃₅₃ (1 μM) at 37° C. for 2 h.Control target cells were pulsed with no peptides. Stimulated SPCs werethen added as effector cells and incubated at 37° C. for 4 h. Onehundred μl of supernatant were collected and radioactivity measured in agamma counter.

FIG. 6 demonstrates that the CTL responses induced by the modifiedpeptides were able to lyse T2 cells loaded with the nativeIL-13Rα2₃₄₅₋₃₅₃. Control non-pulsed EL4-HHD cells were not lysed by theCTLs beyond background levels (shown in FIG. 7). Furthermore, theimmunization with IL-13Rα2-V9 displayed a trend toward higher levels ofCTL reactivity against the EL4-HHD cells pulsed with the nativeIL-13Rα2₃₄₅₋₃₅₃ peptide than other peptides examined, although thedifference was not statistically significant due to the variation withinthe triplicated samples. These data support the previous set of datawith human HLA-A2+ patient derived T cells, in which the modifiedpeptides induced higher levels of anti-IL-13Rα2₃₄₅₋₃₅₃ CTL response thanthe native peptide.

The ability of the same HHD mice-derived CTLs used in FIG. 6 to lyseEL4-HHD-IL-13Rα2 cells was examined in order to evaluate the ability ofthe CTLs to recognize the IL-13Rα2₃₄₅₋₃₅₃ peptide that is naturallyprocessed by cells that endogenously express IL-13Rα2. FIG. 7illustrates that immunization with the IL-13Rα2₃₄₅₋₃₅₃, IL-13Rα2-V9 or-A1V9 induced a specific CTL activity against EL4-HHD-IL-13Rα2 cells.The CTL activities were antigen-specific because control EL4-HHD werenot lysed beyond the background level. Modified peptides IL-13Rα2-V9 and-A1V9 induced higher magnitude of CTL activities in comparison to nativeIL-13Rα2₃₄₅₋₃₅₃ against the EL4-HHD-IL-13Rα cells (p<0.05 at alleffector/target ratios). The in vivo anti-tumor effect of vaccinationswith the IL-13Rα2₃₄₅₋₃₅₃ or modified IL-13Rα2 peptides in HHD micebearing EL4-HHD-IL-13Rα2 tumors is currently being evaluated.

Example 6

This example demonstrates that EphA2 has available HLA-A2-restricted CTLepitopes.

EphA2 is an attractive tumor-associated antigen and a target fortumor-vaccines, as 5 HLA-A2 and 3 DR4 T cell epitopes have beenpreviously identified (Tatsumi et al., 2003). As shown in FIG. 8, 9 of14 human glioblastoma multiforme (GBM) and 6 of 9 anaplastic astrocytoma(AA) cases express high levels of EphA2. In addition, anti-glioma CTLreactivity has been induced in CD8+ cells obtained from HLA-A2+ gliomapatients by stimulation with the EphA2₈₈₃₋₈₉₁ epitope (FIG. 9). Thisresponse was specific for the EphA2₈₈₃₋₈₉₁ epitope because the parallelassay using T2 cells loaded with EphA2₈₈₃₋₈₉₁ demonstrated apeptide-specific response in comparison to the control unloaded T2target (not shown). These data strongly suggest that EphA2₈₈₃₋₈₉₁ canserve as a CTL epitope.

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All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of vaccinating a patient againstglioma, comprising introducing into the patient (i) a peptide comprisingTLADFDPRFV (SEQ ID NO:6) and (ii) a peptide comprising ALPFGFILV (SEQ IDNO:3)under conditions sufficient for said patient to develop a cytoxicT-cell lymphocyte (CTL) response.
 2. A method of vaccinating a patientagainst glioma, comprising introducing into the patient a compositioncomprising (i) a peptide comprising TLADFDPRV (SEQ ID NO:6), (ii) apeptide comprising ALPFGFILV (SEQ ID NO:3), and (iii) a physiologicallyacceptable carrier under conditions sufficient for said patient todevelop a cytoxic T-cell lymphocyte (CTL) response.
 3. The method ofclaim 2 wherein said physiologically acceptable carrier is apharmaceutically-acceptable carrier.
 4. The use of claim 2, wherein saidcomposition further comprises a T-helper epitope.
 5. The method of claim3, wherein said composition further comprises a T-helper epitope.
 6. Theuse of claim 4, wherein the T-helper epitope is a peptide comprising theamino acid sequence TPPAYRPPNAPIL (SEQ ID NO:5).
 7. The method of claim5, wherein the T-helper epitope is a peptide comprising the amino acidsequence TPPAYRPPNAPIL (SEQ ID NO:5).
 8. The method of claim 1 or 2,wherein the glioma is ependymoma, astrocytoma, oligodendroglioma,glioblastoma, or a mixed glioma.
 9. The method of claim 1 or 2, whereinsaid patient is a mammal.
 10. The method of claim 1 or 2, wherein saidpatient is human.
 11. The method of claim 1, wherein the peptide of (i)consists of TLADFDPRV (SEQ ID NO:6).
 12. The method of claim 2, whereinthe peptide of (i) consists of TLADFDPRV (SEQ ID NO:6).
 13. The methodof claim 1, wherein the patient develops a CTL response against gliomacells in the patient.
 14. The method of claim 2, wherein the patientdevelops a CTL response glimoa cells in the patient.